A murine model of hnRNPH2-related neurodevelopmental disorder reveals a mechanism for genetic compensation by Hnrnph1

Mutations in HNRNPH2 cause an X-linked neurodevelopmental disorder with features that include developmental delay, motor function deficits, and seizures. More than 90% of patients with hnRNPH2 have a missense mutation within or adjacent to the nuclear localization signal (NLS) of hnRNPH2. Here, we report that hnRNPH2 NLS mutations caused reduced interaction with the nuclear transport receptor Kapβ2 and resulted in modest cytoplasmic accumulation of hnRNPH2. We generated 2 knockin mouse models with human-equivalent mutations in Hnrnph2 as well as Hnrnph2-KO mice. Knockin mice recapitulated clinical features of the human disorder, including reduced survival in male mice, impaired motor and cognitive functions, and increased susceptibility to audiogenic seizures. In contrast, 2 independent lines of Hnrnph2-KO mice showed no detectable phenotypes. Notably, KO mice had upregulated expression of Hnrnph1, a paralog of Hnrnph2, whereas knockin mice failed to upregulate Hnrnph1. Thus, genetic compensation by Hnrnph1 may counteract the loss of hnRNPH2. These findings suggest that HNRNPH2-related disorder may be driven by a toxic gain of function or a complex loss of HNRNPH2 function with impaired compensation by HNRNPH1. The knockin mice described here are an important resource for preclinical studies to assess the therapeutic benefit of gene replacement or knockdown of mutant hnRNPH2.


Generation of human iPSCs bearing hnRNPH2 mutations
Genetically modified AN1.1 iPSC lines were generated using CRISPR-Cas9 technology. Briefly, for each modification, unmodified AN1.1 iPSCs were pretreated for 1 hour in StemFlex (Thermo Technologies) coated 96-well plates. Clones were screened for the desired modification via targeted deep sequencing on a Miseq Illumina sequencer as previously described (5). Samples were demultiplexed using the index sequences, fastq files were generated, and NGS analysis of clones was performed using CRIS.py (6). Correctly modified clones were identified, expanded, and sequence confirmed. Cell identity was authenticated using the PowerPlex® Fusion System  Table 5.

Differentiation of iPSC-derived neurons
iPSCs were differentiated into cortical neurons with a two-step protocol (pre-differentiation and maturation) as previously described (7). When iPSCs reached 70-80% confluence, cells were washed twice with DPBS and dissociated with Accutase (STEMCELL Technologies) and collected cells were filtered using a cell strainer (STEMCELL Technologies). Millipore). For visualization, the appropriate host-specific Alexa Fluor 488, 555, or 647 (Invitrogen) secondary antibody was used. Slides were mounted using Prolong Gold Antifade Reagent with DAPI (Life Technologies). Images were captured using a Leica TCS SP8 STED 3X confocal microscope (Leica Biosystems) with a 63x objective. Fluorescent images were subjected to automated compartmentalization analysis using CellProfiler software (Broad Institute). Cells were segmented using DAPI and eIF3η channels to identify the nucleus and cytoplasm. Integrated intensity of nucleus, cytoplasm, and cells were measured. Percent cytoplasmic signal was calculated with the integrated cytoplasmic signal over the integrated cell signal.

Immunoprecipitation and Western blot analysis in cell lines
Cell lysates were prepared by lysing cells in buffer containing 20 mM phosphate buffer pH 7.5, 150 mM NaCl, 0.2% Triton X-100, and 10% glycerol with complete protease inhibitor cocktail (Clontech Laboratories). Cells were incubated on ice for 20 minutes before centrifugation at 14,000 rpm at 4°C. The resulting supernatant was pre-treated with EZview Red Protein A agarose beads (P6486; Sigma) for 45 minutes to reduce the likelihood of nonspecific binding to the agarose, and the beads were removed. EZview Red Anti-FLAG M2 agarose beads (F2426; Sigma) were then added to the pre-treated lysates and incubated at 4°C for 2 hours. The agarose beads were washed three times with buffer above to remove any remaining nonspecific binding. Samples were eluted with FLAG peptide (F3290; Sigma) at a final concentration of 100 μg/ml for 30 minutes at vortex setting 5 (Scientific Industries) at 4°C. Samples were boiled in 1x LDS sample buffer (Thermo Fisher). Samples were resolved by electrophoresis on NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen). Gels were transferred to nitrocellulose using an iBlot 2 gel transfer device (Thermo Fisher) and blocked in 5% BSA. Primary antibodies used were rabbit polyclonal anti-FLAG (F7425; Sigma) and mouse monoclonal anti-Kapβ2 (ab10303; Abcam). Blots were subsequently incubated with IRDye fluorescence antibodies (LI-COR) and protein bands were visualized using the Odyssey Fc system (LI-COR) and Image Studio (LI-COR). Bands were quantified by densitometry in ImageJ (NIH). The full, uncut gels are included in the Supplemental Material.
Pulldown assays for Kapβ2 binding to immobilized GST-hnRNPH2 peptides E. coli (BL21) transformed with pGEX-4TT3 plasmids expressing GST-hnRNPH2 proteins were grown in 35 ml LB with 100 μg/ml ampicillin to OD600 0.6. Protein expression was then induced with 0.5 mM isopropyl-β-d-1-thiogalactoside (IPTG) for 5 hours at 37°C. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 15% glycerol, and protease inhibitors), lysed by sonication, the lysate centrifuged, and supernatant containing GST-hnRNPH2 proteins added to Glutathione Sepharose 4B (GSH; GE Healthcare) beads. The beads with immobilized GST-hnRNPH2 proteins were washed with lysis buffer. 50 μl beads containing ~60 μg immobilized GST-hnRNPH2 proteins were incubated with 8 μM Kapβ2 in 100 μl total volume for 30 minutes at 4°C and then washed three times with 1 ml lysis buffer. Proteins bound on the beads were eluted by boiling in SDS sample buffer and visualized by Coomassie staining of SDS-PAGE gels.

Generation of Hnrnph2 mutant and knockout mice
For the R206W mutation, two conserved C nucleotides at positions 833 and 835 were substituted with T and G, respectively (c.833 C > T and c.835 C > G). For the P209L mutation, the C nucleotide at position 842 was substituted with T (c.842 C > T). gRNA was in vitro transcribed using MEGAshortscript T7 kit (Life Tech Corp; AM1354), and the template PCR amplified using the following primers:

Mendelian inheritance and survival up to 8 weeks
All pups born and genotyped (samples collected from live pups at P2-P7 and from pups found dead before P2-P7 sample collection) in the colonies from April 2018 to March 2021 were included in calculation of genotype ratios. All pups born and genotyped during this time were also included in survival analyses, except for mice used in cohort 2 (audiogenic seizure cohort) and cohort 3 (µCT and imaging cohort).

Behavioral phenotyping and long-term survival
Experimental cohort 1, consisting of male hemizygous mutants or KOs, female heterozygous mutants or KOs, and WT littermate controls, were first subjected to an observational test battery at 8 weeks old. This was followed by more specific and sensitive tests of motor and sensory function at 8-9 weeks and 10-12 weeks, respectively. These mice were also weighed weekly from 3 to 8 weeks, then again at 6 months and every 6 months thereafter and followed for survival.
A slightly modified protocol of the EMPReSS (European Mouse Phenotyping Resource for Standardized Screens) version of SHIRPA (SmithKline Beecham, Harwell, Imperial College, Royal London Hospital, Phenotype Assessment) level 1 observational test battery was used (9). Briefly, mice were observed undisturbed in a clear viewing jar for activity, tremor, palpebral closure, coat appearance, skin color, whisker appearance, lacrimation, defecation, and urination. Mice were then moved to an arena and the following parameters scored: transfer arousal, locomotor activity, gait, pelvic elevation, tail elevation, startle response, touch escape and righting reflex. Thereafter, mice were held by the tail and scored for positional passivity, trunk curl, limb clasping, and visual placing. After placement on a wire mesh grid, mice were assessed for corneal reflex, pinna reflex, whisker orienting reflex, toe pinch response, and negative geotaxis. Lastly, contact righting response when place in a tube and rolled upside down was tested, and any evidence of biting and excessive vocalization noted. The data were quantified using a binary scoring system as previously described (10). A normal behavior received a score of 0 and an abnormal behavior received a score of 1, enabling a global abnormality score to be determined for each mouse, with a higher score corresponding to a greater degree of abnormality. In addition, scores were also generated for specific functions including motor, sensory, neuropsychiatric, and autonomic function (11).
Rotarod analysis was performed on an accelerating rotarod apparatus (IITC Life Science) using a 2-day protocol. Mice were trained on the first day with one session set at 4 rpm for 5 minutes. The following day, rotation speed was set to accelerate from 4 to 40 rpm at 0.1 rpm/s, mice were placed on the apparatus, and the latency to fall was recorded for four separate trials per mouse. Mice were given a 15-minute rest period between each trial. Grip strength was measured using a grip strength meter (Bioseb) as grams of force for all 4 paws for each mouse in six repeated measurements. The beam walking test was performed using a 2-day, multibeam protocol (12). Briefly, on day 1 mice were trained to walk across an elevated 12-mm square beam to reach an enclosed goal box. On day 2, mice received one trial each on a 12mm square beam, a 6-mm square beam, and a 12-mm round beam, and latency to cross, number of hind paw slips, and number of falls recorded. A custom neurological scoring system was also used, where a score of 0 was given if the mouse was unable to traverse the beam in 60 s, 1 if a mouse traversed the entire beam by dragging itself with its front paws (hind paws remain in contact with the side of the beam at all times), 2 if a mouse was able to traverse the beam with some hind paw stepping on top of the beam before starting to drag itself with its front paws, 3 if a mouse was able to traverse the entire beam with hind paw stepping, but placed its hind paws on the side of the beam at least once (no dragging with front paws), and 4 if a mouse was able to traverse the entire beam with hind paw stepping and never placing its hind paws on the side of the beam. In the wire hang test, mice were placed onto a wire cage top, which was then inverted and elevated above a clean cage, and latency to fall (up to 120 s) recorded. For gait analysis, the front and hind paws of each animal were dipped in red and blue paint (watersoluble and non-toxic), respectively. The animal was then placed in a 70-cm long tunnel lined on the bottom with Whatman filter paper, the entrance sealed, and animal allowed to walk through one time. Footprints were scanned and analyzed with Image J for stride length, fore-and hind base width, and overlap (13).
Experimental cohort 2, consisting of male hemizygous mutants or KOs, female heterozygous mutants or KOs, female homozygous mutants or KOs, and WT littermate controls, were tested for audiogenic seizure susceptibility in a clear acrylic box (30 x 30 x 30 cm), with a 6" red fire bell mounted to the underside of a removable lid, and connected to a standard GraLab timer. The bell consistently produced 120-125 dB sound as measured from inside the closed box using a digital sound level meter. At P21, mice were removed from their home cage one by one just before testing, put into a clean holding cage, and moved to the testing room.
Mice were then transferred to the audiogenic seizure chamber and allowed to explore the box for 15 s before the bell was turned on for 60 s. The intensity of the response (seizure severity score) was categorized as 0 for no response or slight startle, 1 for wild running, 2 for clonic seizures, 3 for tonic seizures, and 4 for respiratory arrest (14).
Experimental cohort 4, consisting of hemizygous R206W males and WT littermate controls, were subjected to a battery of tests over 4 weeks to assess learning and memory, emotional behaviors, and social behaviors. To reduce any potential carryover effects, tests were run in the order of least invasive to most invasive, based on the sensitivity of tests on previous handling, and stress induced by each test (15). Tests were conducted with a 1-2 day interval, which has been shown to have little impact on performance compared to 1 week inter-test intervals (16). Starting at 8 weeks old, experimentally naïve mice were tested for anxiety in the elevated plus maze, anxiety and locomotion in the open field test, visual recognition memory in the novel object recognition test, spatial working memory in the Y maze spontaneous alternation test, social preference in the three-chamber social interaction test, spatial learning and memory in the Morris water maze, and repetitive, compulsive-like behavior in the marble burying test.
For all tests, mice were moved to the test room, kept behind a room divider, and allowed to acclimatize undisturbed for 30 minutes before starting tests. At the end of a test, mice were placed in a holding cage until all mice from a home cage completed testing, before being returned to the home cage. During all tests, the investigator remained out of sight behind a room divider. Behavioral parameters were recorded and analyzed using ANY-maze automated activity monitoring system and software (v7.2). To facilitate automated tracking and reduce stress on the animals, all tests were conducted under low (30-50 Lux), indirect lighting. Mazes, objects (novel object recognition test) and wire cages (three-chamber social interaction test) were thoroughly cleaned with 70% vol/vol ethanol after each test and allowed to dry before starting the next test.
In the elevated plus maze (Stoelting), mice were placed in the center facing an open arm away from the investigator and allowed to explore the maze undisturbed for 5 minutes (17).
Parameters including total distance traveled, mean speed, open and closed arm entries and time, and center entries and time were recorded and analyzed for the total test time, as well as temporally across the session to assess any potential differences in habituation to novelty and aversive learning (18,19). The animal's entire area was used to score arm entries and exits, with at least 80% of the animal needed for an arm entry to occur, and mice were considered to be in the center zone if not in any arm. Mice that fell off the maze were excluded from the

analysis.
The open field test was run according to the protocol by Seibenhener and Wooten (20).
Briefly, mice were placed in the center of a 40 cm x 40 cm open field arena (Stoelting) facing away from the investigator and allowed to explore undisturbed for 20 minutes. Parameters including total distance traveled, mean speed, time in the center zone (defined as the center 24 cm x 24 cm of the maze) were recorded and analyzed for the total test time, as well as temporally across the session to assess any potential differences in habituation to novelty (21).
The animal's center was used to score zone entries and exits.
The novel object test protocol used was based on those published by Leger et al. (22) and Lueptow (23) and included a familiarization phase and test phase. As the test was In the Y maze (Stoelting), mice were placed in a distal part of an arm, facing away from the investigator and allowed to explore the maze freely for 8 minutes. Parameters including total distance traveled, mean speed, number of entries and time spent in each arm and the maze center were recorded and analyzed for the total test time. The animal's entire area was used to score arm entries and exits, with at least 80% of the animal needed for an arm entry to occur, and mice were considered to be in the center zone if not in any arm. A spontaneous alternation was defined as consecutive entry into 3 different arms on overlapping triplet sets, and the percentage of spontaneous alternations was calculated as the number of spontaneous alternations divided by the total number of arm entries minus 2, multiplied by 100 (24). Mice that climbed out of the maze were excluded from the analysis.
The three-chamber social interaction test consisted of a pre-test, followed the next day by a social preference test and social novelty test, with an inter-test interval of 5 minutes (25). In the pre-test, mice were placed in the center chamber of the sociability cage (Stoelting) facing away from the investigator and allowed 10 minutes to freely explore the cage, which contained crumpled paper balls inside wire enclosures placed in the right and left chambers of the cage.
The next day, the 2 wire cages contained either a wooden block to serve as the non-social stimulus, or an unfamiliar age-, sex-, and background strain-matched WT mouse to serve as the social stimulus. The test mouse was placed in the center chamber of the cage facing away from the investigator and allowed to explore the cage undisturbed for 10 minutes. At the end of the test, the test mouse was removed and placed into a holding cage and the wooden block was replaced by an unfamiliar age-, sex-, and background strain-matched WT mouse to serve as the novel social stimulus. The mouse used as the social stimulus during the social preference test was kept in the wire cage and served as the known social stimulus during the social novelty test. After 5 minutes, the test mouse was again placed in the center chamber of the sociability cage facing away from the investigator and allowed to explore the cage undisturbed for 10 minutes. Parameters including total distance traveled, mean speed, and time investigating each stimulus were recorded and analyzed. Stimulus investigation was defined as the animal's head being within 25 mm of the wire cage and oriented toward the stimulus (orientation angle of 60°).
A social preference index was calculated by subtracting the time spent investigating the nonsocial stimulus from the time spent investigating the social stimulus and dividing the result by the time spent investigating the social stimulus plus the non-social stimulus. A social novelty index was calculated by subtracting the time spent investigating the known social stimulus from the time spent investigating the novel social stimulus and dividing the result by the time spent investigating the known social stimulus plus the novel social stimulus (25).
The Morris water maze protocol was modified from that of Vorhees and Williams (26). A blue circular plastic pool was used with a diameter of 120 cm and depth of 81 cm along with an adjustable height circular platform (grey for cued trials, clear for training trials) with a diameter of 10 cm (MazeEngineers). The pool was filled with water to approximately 20 cm from the top and allowed to equilibrate to room temperature (approximately 20°C) for at least 2 days. To facilitate automated tracking and reduce visibility of the platform during training trials, 3 bottles of nontoxic white tempera paint were added to the pool. A cued test was first performed during which the pool was surrounded by black room dividers to eliminate any distal room cues and the platform was visible (height was adjusted to just above the surface of the water and a red plastic flag was attached to the grey platform to improve visibility). The cued test consisted of 4 trials performed on a single day, with an inter-trial interval of 10-15 minutes (mice were run in blocks of 10 and all mice completed a trial before the next trial was started). The position of the visible platform was moved for each trial (southeast, northeast, southwest, northwest) and the starting position was alternated between north and west. Mice were gently placed in the water facing the pool wall and given 60 seconds to find and climb on to the visible platform. Mice that failed to find the platform were gently picked up and placed on the platform. Once on the platform, they were allowed to remain there for 15 seconds. At the end of each trial, mice were returned to a heated holding cage while waiting to start the next trial and returned to the home cage at the completion of the 4 trials. Two days later, the mice were subjected to 4 days of training, consisting of 4 trials per day, with an inter-trial interval of 10-15 minutes (mice were run in blocks of 10 and all mice completed a trial before the next trial was started). During training trials, only 1 black room divider was used to hide the investigator from view during the test. A black and white stripe poster on the room divider, as well as objects in the testing room around the pool (e.g., two lamps, wall cabinet) served as distal spatial cues. A clear platform was submerged 1-2 cm below the surface of the water and was not visible under the water with white paint added. A set of semi-randomly selected distal start positions were used, with the platform remaining in the southwest quadrant as previously described (26). Mice were gently placed in the water facing the pool wall and given 60 seconds to find and climb on to the hidden platform. Mice that failed to find the platform were gently picked up and placed on the platform.
Once on the platform, they were allowed to remain there for 15 seconds. At the end of each trial, mice were returned to a heated holding cage while waiting to start the next trial and returned to the home cage at the completion of the 4 trials. After 4 days of training, mice were tested for spatial memory in a single probe trial, during which the platform was removed from the pool. Mice were gently placed in the water facing the pool wall at a location not used during training, directly across from the previous location of the platform (northeast), and removed after 30 seconds. Parameters including total distance traveled, mean swim speed, time in each quadrant, latency to reach the platform (cued and training trials), percentage of time in the thigmotaxis zone (within 10 cm of the pool wall), cumulative distance from hidden platform (training trials), platform location crossings (probe trial), and mean distance from platform location (probe trial) were recorded and analyzed. Data for the training trials are averaged across 4 trials per day and plotted as block means. The animal's center was used to score zone entries and exits.
The marble burying test was performed according to the protocol by Angoa-Perez et al. (27). Standard polycarbonate rat cages were filled with fresh mouse bedding to a depth of 5 cm and the bedding surface leveled. Twenty standard glass toy marbles of assorted styles and colors were placed gently on the surface of the bedding in 5 rows of 4 marbles each. A single mouse was placed in a cage away from the marbles, the cage was covered with a filter-top lid, and the mouse allowed to remain in the cage undisturbed for 1 hour. After the test, the mouse was removed, taking care not to move any marbles, and returned to its home cage. The number of marbles buried (at least two-thirds of surface area covered by bedding) was counted.

EEG implantation
EEG/EMG headstage (Model 8431-SM, Pinnacle Technology) implantation was completed according to the manufacturer's instructions unless otherwise noted. In brief, mice were anesthetized using isoflurane vapors at 3% and maintained during implantation at 1.5%. A small midline incision was made, exposing the skull. Six holes were made in the skull using a 23gauge needle; following this, each hole had a screw with a wire lead attached placed inside.
Dental cement (DuraLay, Reliance Dental Manufacturing) was used to cover the screws, leaving the wires exposed. The headstage was then placed on top of the dried dental cement and EMG leads were inserted into the nuchal muscles via a pocket created using forceps. Headstage wires were then connected to the lead wire from the screw using wire glue (Anders Products) and allowed to dry completely before covering them with dental cement. Animals were then given a post-operative analgesic injection (meloxicam, 2 mg/kg) and mush food for the next three days to ease recovery. Animals were given at least one week to recover prior to data collection.

EEG data collection and analysis
EEG/EMG collection was synchronized with video recording using Pinnacle Seizure Acquisition Software (v2.1.0). Data was obtained using the Pinnacle Acquisition System with a 8406-SE31M pre-amplifier at a sampling rate of 2000 Hz with a high and low bandpass filter at 0 and 500 Hz, respectively. Representative 1-hour time spans were selected in an unbiased manner (12am-1am and 6:30am-7:30am for the dark and light phases, respectively) and were analyzed blind to genotype for spectral power and epileptiform activity. All analyzed data was recorded after at least 2 hours of acclimation to the recording arena and connection to the wiring tether.
Electrical activity was converted to frequency using a fast Fourier transformation (FFT) algorithm using the Hann method. Pinnacle Sirenia Seizure Pro (v2.2.5) software was used for spectral power analysis at individual frequencies and delta (0.5-4 Hz), theta (5-Hz), alpha (9)(10)(11)(12)(13) Hz), and beta (14-30 Hz) bands. Spectral power data were used in combination with video recordings to rule out motion-based artifacts and to manually quantify the percentage of time spent in epileptiform activity. Spikes that were greater than 2x the baseline amplitude and <200 ms in duration and showing polyspikes (defined as spikes crossing baseline more than two times) were considered epileptiform. The "time spent" satisfying criterion was summed across the entire hour and calculated as the percentage of time spent exhibiting the phenotype. The lambdoid lead EEG waveform and video from 12am to 1am was watched and scored to detect any absence-like (behavioral arrest during EEG spiking) or motor (behavioral changes similar to myoclonic jerks, tonic-clonic action, wild running, or postural loss) seizures.

In vivo MRI and µCT
Experimental cohort 3, consisting of male hemizygous mutants or KOs, female heterozygous mutants or KOs, and WT littermate controls, were imaged at the Center for In Vivo Imaging and Therapeutics at St. Jude Children's Research Hospital using micro-computed tomography (µCT) and magnetic resonance imaging (MRI) at 6 and 24 weeks of age. The µCT was performed on a Siemens Inveon PET/CT system (Siemens) at 88-µm resolution, and the MRI was performed on a Bruker Clinscan 7T MRI system (Bruker Biospin MRI GmbH). MRI was acquired with a mouse brain surface receive coil positioned over the mouse head and placed inside a 72-mm transmit/receive coil. After the localizer, a T2-weighted turbo spin echo sequence with variable flip-angle echo trains was performed in the coronal orientation (TR/TE = 2500/114 ms, matrix size = 192 × 192 x 104, resolution = 0.12 x 0.12 x 0.12 mm, number of averages = 4). Prior to scanning, mice were anesthetized in a chamber (3% isoflurane in oxygen delivered at 1 L/min) and maintained using nose-cone delivery (1-2% isoflurane in oxygen delivered at 1 L/min).
Animals were provided thermal support using an inbuilt electronic heating pad (µCT) or a heated bed with warm water circulation (MRI) and a physiological monitoring system to monitor breath rate. After imaging, animals were allowed to recover on a heating pad.
Morphometric analysis was performed on the µCT images to identify group differences in skull shape. Linear measurements of 11 key craniofacial parameters (28) were performed manually on µCT slices using Inveon Research Workplace software (IRW 4.2, Siemens). This was followed by automated imaged-based shape analysis using a population-level atlas of the Mus musculus craniofacial skeleton (29). Briefly, the head was extracted from the whole-body µCT images using an iterative search and best-match algorithm. The µCT atlas (https://github.com/muratmaga/mouse_CT_atlas) was then aligned to native space images using a first pass affine transform, followed by a non-linear warping. The calculated transform was then applied to a set of 51 previously identified landmarks and the coordinates for the landmarks in native space were extracted. Processing steps were performed using the ANTS software package (https://github.com/ANTsX/ANTsPy). All alignment results were visually inspected by at least 2 raters. The Euclidean distance between each point was calculated and used for subsequent analysis. First, we performed pairwise comparisons of linear distances between all 51 landmarks. Next, we performed Euclidean distance matrix analysis (EDMA), a geometric morphometric approach enabling the quantification and comparison of shape in three dimensions (30). For global EDMA analysis all 51 landmarks were included, whereas the regional EDMA analysis was performed on a subset of landmarks that summarize regions with specific embryonic tissue origins, further divided into anatomically relevant subsets including palate, midface, and nasal regions (31). To account for overall difference in size, both the global and regional EDMA analyses were scaled to centroid size (calculated as the square root of the sum of squared distances of all landmarks from their centroid), which is a common proxy for overall size in geometric morphometric analyses (32).
Brain parcellation and volumetrics were performed to investigate group differences in total and regional brain volumes. We used the DSURQE atlas (33), which contains 336 cortical, white matter, subcortical, and CSF defined regions. The DSURQE anatomical image was first downsampled to 120-μm isotropic resolution to satisfy the Nyquist criteria of our image resolution and reduce computational time for fitting. The acquired T2 images were preprocessed, including skull-stripping and intensity normalization. The images were then aligned to the atlas by a first-pass affine registration, followed by a non-linear warping. The inverse warping was applied to the labeled atlas to bring all labeled areas into native space. All image processing steps were performed using the AFNI software package (https://afni.nimh.nih.gov/). The volume (number of voxels times native resolution) of each labeled area from the atlas was extracted for subsequent analysis. The results of the inverse warping were quality checked by visual inspection by at least 2 raters. Cases with poor alignment (17 out of a total of 140) were removed from the final volumetric analysis.

Mouse histology and immunofluorescence
For confirmation of hydrocephalus, mice were anesthetized by isoflurane inhalation and transcardially perfused with 10% neutral buffered formalin (NBF) (mice flagged for domed heads) or postfixed in 10% neutral buffered formalin (mice found dead). Heads were decalcified, paraffin-embedded in the coronal plane, 10 4-µm step sections (every 50 µm) cut, stained with hematoxylin and eosin (H&E), and reviewed by a veterinary pathologist.
Brains from experimentally naïve male hemizygous mutants or KOs and male WT littermate controls were harvested at 8 weeks (Hnrnph2 R206W and KO) or 3 weeks (Hnrnph2 P209L ) of age for histology and immunofluorescence. Briefly, mice were anesthetized by isoflurane inhalation and transcardially perfused with 10% NBF, the brain dissected from the skull and cut in half on the sagittal plane, processed for paraffin embedding, and cut at 10 µm.

Primary cortical neuron culture
Primary culture of cortical neurons was prepared from P1 mice as previously described (38).  and GFP, a tilescan of 592.92 µm x 592.92 µm was taken centered around the soma, with a zstep size of 0.3 µm for a total z-stack size between 15 µm and 40 µm per neuron. Acquisition of the GFP channel was performed using a 488-nm laser at 50% power with 100-ms exposure.

Magnetofection of primary cortical neurons
Stitching was performed automatically in Nikon Elements.

Image analysis of dendritic arborization and spines
Stitched images were imported into Imaris (Bitplane v9.9.1), and using the in-built Filaments module, dendrites and dendritic spines originating from the centered soma in each image were segmented. Any dendrite originating from the cell body was considered as a primary dendrite.
A dendritic branch point was determined when the original dendrite bifurcated into two or more daughter trees. For assigning branch level, the lowest level was given to the primary dendrites and levels were increased when a branch point was reached. Dendritic full branch depth was calculated by the sum of dendritic full branch points of a given neuron and dendritic full branch level was the maximum value reached by any primary tree on a given neuron. For Sholl analyses, Sholl radii originating from the centroid of the soma were increased at 1-μm intervals. The Sholl intersection profile was obtained by counting the number of dendritic branches at each given distance from the soma and/or averaging them across the entire neuron. Morphometric measurements for dendrite full branch depth, dendrite full branch level, dendrite length, number of Sholl intersections, total number of spines, and spine density (per 10 µm) of the segmentation were extracted and plotted in GraphPad Prism 9.

RNA sequencing
Total RNA was extracted with an RNeasy Universal Plus Mini kit (Qiagen; 73404). A sequencing library was prepared with a TruSeq Stranded Total RNA Kit (Illumina) and sequenced with an Illumina HiSeq system with 100-bp read length. Total stranded RNA sequencing data were processed by the internal AutoMapper pipeline. Raw reads were first trimmed (Trim-Galore v0.60), mapped to mouse (GRCm38) or human (hg38) genome assembly (STAR v2.7) and then the gene level values were quantified (RSEM v1.31) based on GENCODE annotation (vM22).
We obtained the TPM (transcript per million) counts for genes with TPM greater than one in at least one sample and the gene expression analysis was performed with non-parametric ANOVA using Kruskal-Wallis and Dunn's tests on log-transformed TPM counts between three replicates of each experimental group, implemented in Partek Genomics Suite v7.0 software (Partek). The expression of a gene was considered significantly different if P < 0.05 and log2FC > 0.5 in at least one of the group comparisons. The calculated z-scores of significantly differential expressed genes or log2Rs were plotted using hierarchical clustering in a heat map, using correlation distance measure, implemented in Spotfire v7.5.0 software (TIBCO). For alternative splicing analysis, the aligned and sorted BAM files after STAR alignment were used for AS analysis using rMATS (v4.0.2) (50). A3SS, A5SS, SE, RI, and MXE events were evaluated.
Significant AS events were identified while average coverage >10 and delta percent spliced in (ΔPSI) > 0.1.

Statistics
Significant differences from expected Mendelian inheritance ratios were determined by chisquare tests. The log-rank (Mantel-Cox) test was used to determine significant differences between survival curves and hazard ratios computed by a log-rank approach. Probe trial data were analyzed by unpaired t test (mean speed, mean distance from platform location, % time in the thigmotaxis zone, platform location crossings) and two-way ANOVA (quadrant, genotype, quadrant x genotype interaction) followed by Sidak's multiple comparisons test to compare mutants and controls. Y maze, novel object recognition and three chamber social interaction data were analyzed by unpaired t tests. In the Y maze, the correlation between total arm entries and % spontaneous alternations were evaluated by Pearson's correlation.

Study approval
All studies were approved by the St. Jude Children's Research Hospital institutional review committee on animal safety.